Although our general knowledge about cGMP formation and its cellular effects has substantially increased in the last years, critical gaps remain. These gaps mostly concern the mechanisms and consequences of subcellular cGMP compartmentation, and the distal signalling pathways that mediate the positive vs negative effects of cGMP on cell growth and survival. These questions are particularly difficult to address, but the answers are needed to understand and potentially improve repair mechanisms in the diseased state, such as cardiac and vascular remodelling, ischaemic heart disease, neuronal degeneration/regeneration, and hearing loss. In the FOR 2060 research consortium, investigators from different disciplines share expert knowledge about cGMP signalling in the cardiovascular and nervous system, unique genetic mouse models, and sophisticated optical and functional methods to answer these questions. Of the seven projects proposed for the second funding period (2016-2018), three projects investigate cGMP signalling in the cardiovascular system (project 3, project 4, and project 5), three projects focus on the nervous/neurosensory system (project 6, project 8, and project 10), and one project studies both vascular and neuronal cGMP signalling (project 1).
One of the major questions of our consortium concerns the subcellular compartmentation of cGMP formation and signalling. NO-GC is thought to generate global cytoplasmic cGMP signals, whereas transmembrane pGCs (e.g., GC-A, GC-B, and GC-C) possibly establish local membrane-associated cGMP microdomains. Moreover, the observation of distinct effects of ANP (via GC-A) and CNP (via GC-B) on myocyte calcium homeostasis or contractile functions suggests that even different pGCs generate different cGMP microdomains (Frantz et al. 2013). The aim of project 1 (Feil/Feil) is to improve our understanding of global vs local cGMP signalling in the control of cell death and survival in mammals, with a focus on the interplay of cGMP and remodelling processes in vascular smooth muscle cells (VSMCs) and dorsal root ganglion (DRG) neurons. In the first funding period, transgenic cGMP sensor mice expressing the FRET-based cGi500 sensor globally in all tissues or selectively in specific cell types were generated and characterized (Shuhaibar et al. 2015; Thunemann et al. 2014; Thunemann et al. 2013). Using primary cells and tissues from our cGMP sensor mice, we have begun to visualize cGMP signals in VSMCs and DRG neurons in real time and to correlate them with physiological and pathophysiological cell growth. These studies will now be extended to analyse the in vivo relevance of distinct cGMP signals in the context of vascular remodelling during atherosclerosis and restenosis, for cGMP-mediated axon branching during embryogenesis, and for cGMP-mediated pain processing and axonal regeneration. Project 1 collaborates with other FOR 2060 projects to study the role of NO-GC for VSMC growth (with Friebe, project 3), to develop new biosensors for cGMP/cAMP imaging (with Nikolaev, project 4), to test the existence of mitochondrial cGMP in cardiomyocytes (with Lukowski/Ruth, project 5), and to monitor cGMP in living tissue preparations of healthy and diseased cochlea (with Rüttiger/Knipper, project 8) and dorsal root ganglia (with Schmidt, project 6, and Schmidtko, project 10), as well as in cultured dentate granule cells (with Schmidt, project 6).
Within the cardiovascular system, most cell types (vascular smooth muscle and endothelial cells, fibroblasts, cardiac myocytes) coexpress three different cGMP-generating receptors: NO-GC, GC-A, and GC-B. Projects 2 to 5 of the consortium dissect the specific functions and downstream effectors of these cGMP-generating receptors in the context of cardiovascular cell growth and survival.
In the first funding period, project 2 (Mergia/Koesling) and project 3 (Friebe) have characterized the role of cGMP in the regulation of vascular tone and remodelling of VSMCs. These projects take advantage of different genetic mouse models with global or conditional, smooth muscle-restricted deletion of specific NO-GC isoforms, GC-A, or GC-B, to dissect possible differences in the signalling pathways and vascular effects of the respective ligand/receptor pairs. The aim of project 2 was to study the role of NO-GC in vascular remodelling, particularly the interplay of NO/cGMP with angiotensin II signalling. This project will not be continued in the second funding period of the FOR 2060. A final report will be provided separately. Project 3 focused on the impact of NO/cGMP signalling on cyclic nucleotide crosstalk and on vascular remodelling processes in aorta and lung vessels. These studies revealed a complex NO/cGMP-dependent regulation of the cGMP-inhibited, cAMP-hydrolyzing PDE3 (with Nikolaev, project 4), and an increased pulse wave velocity in the aorta of global NO-GC knockout mice associated with disturbed collagen organization (with Lukowski/Ruth, project 5). One exciting novel finding during the first funding period was that NO-GC is strongly expressed in lung pericytes. Therefore, the project will now focus on the specific role of pericyte NO-GC for lung physiology and fibrosis using cell type-specific NO-GC mutants and lineage tracing strategies (Groneberg et al. 2013). These studies will also include collaborative experiments to monitor cGMP signals in living pericytes (with Feil, project Z) and to test the role of potential cGMP effectors in pericytes, such as cGMP-dependent protein kinases (with Feil/Feil, project 1), BK channels, or CRP4 (with Lukowski/Ruth, project 5).
Project 4 (Nikolaev) and project 5 (Lukowski/Ruth) investigate the protective roles of cGMP in cardiac hypertrophic remodelling and ischaemic pre-/postconditioning. The cardiac pathological response to sustained pressure overload involves myocyte hypertrophy and dysfunction along with interstitial changes such as fibrosis and altered capillary density. These changes are orchestrated by mechanical forces and factors secreted between cells. Among these factors are the natriuretic peptides ANP, BNP (both released from cardiomyocytes), and CNP (secreted from fibroblasts). Interestingly, activation of NO-GC decreases myocardial contractility, while activation of pGCs with either ANP or CNP can lead to opposing functional effects, i.e. an increase in contractility of the failing heart. Based on these observations, project 4 studies the differential effects of cGMP pools produced by NO-GC and various pGCs (GC-A, GC-B) in healthy and diseased cardiomyocytes. In the first funding period, transgenic mice have been generated and characterized that allow visualization of cGMP in the cytosol of cardiomyocytes (Götz et al. 2014) as well as in functionally relevant compartments such as T-tubular membranes. Correlation of subcellular cGMP dynamics in various microdomains with the development of pathophysiological cardiomyocyte growth uncovered a previously unknown increase of catecholamine-stimulated cardiac contractility by ANP in the setting of compensated cardiac hypertrophy (Perera et al. 2015). The results of this project indicate that cardiac disease is associated with cGMP microdomain remodelling, which results from dynamic changes of cGMP/cAMP crosstalk due to PDE and receptor redistribution. In the second funding period, project 4 will continue to analyse changes of submembrane cGMP signalling in hypertrophy and heart failure using two disease models – early compensated cardiac hypertrophy and heart failure following myocardial infarction in mice (with Lukowski/Ruth, project 5). In collaboration with other FOR 2060 projects, the role of NO-GC (with Friebe, project 3) and GC-B (with Schmidt, project 6) for the establishment of cardiomyocyte cGMP microdomains will be studied. Importantly, the findings on cGMP dynamics obtained in single isolated cardiomyocytes will now be further verified using a newly established FRET imaging setup in intact Langendorff perfused hearts. The technical expertise and tools provided by this project do also perfectly complement the imaging-based projects 1 and Z, and strengthen the overall focus of the research unit regarding imaging technology.
The role of the cGMP/cGKI pathway in myocardial protection from ischaemia by preconditioning has been intensively investigated. The infarct-reducing and cardioprotective effects of hormones (ANP, NO) and drugs (PDE5 inhibitors) enhancing this pathway have been attributed to a preconditioning-like effect that increases survival of cardiomyocytes after ischaemia/reperfusion (I/R) injury. However, the downstream pathways mediating the cytoprotective effect of cGMP/cGKI are largely unknown. One potential target of cGKI is the Ca2+-activated K+ channel of the BK type in the inner mitochondrial membrane (mitoBK). MitoBK significantly contributes to mitochondrial K+ uptake, depolarizes the mitochondrial membrane and attenuates mitochondrial Ca2+ overload. Project 5 combines genetic mouse models, electrophysiology, pharmacology, and biochemistry to investigate the role of cGMP generators (NO-GC, GC-A, GC-B) and cGMP effectors (cGKI and mitoBK) in ischaemic pre- and postconditioning. In the previous funding period, it could be shown that BK channels indeed afford protection against I/R injury (Soltysinska et al. 2014). Using the Cre/loxP recombination system, mice with cardiomyocyte-restricted deletions of the BK channel, cGKI (Methner et al. 2013b), or NO-GC were generated and characterized in a cardiac I/R injury model. In collaboration with Friebe (project 3) and Nikolaev (project 4) it was shown that the PDE5 blocker sildenafil does not interfere with the pathogenic actions downstream of the angiotensin II receptor in cardiomyocytes (Straubinger et al. 2015). These and other collaborations (with Feil/Feil, project 1 and project Z) will be continued in the second funding period to study the dynamics of cytosolic/mitochondrial cGMP signals in cardiomyocytes upon an ischaemic insult, and to analyse whether cardioprotection by NO-GC and BK channels is mediated via a canonical cGMP pathway that involves cGKI in cardiomyocytes and/or by alternative non-cardiomyocyte-based mechanisms.
Neuronal cGMP signalling has critical functions during embryonic development and possibly also in the adult organism, where it might promote axonal regeneration. Project 6 (Schmidt) and the new project 10 (Schmidtko) study the cellular and temporal expression patterns of the neuronal cGMP signalling system at different developmental stages, its regulation, downstream effectors, and role in pain processing and regeneration after peripheral nerve injury. Previous work of project 6 has demonstrated that the CNP/GC-B/cGKI pathway is essential for sensory axon branching of DRG neurons at the dorsal root entry zone of the spinal cord during embryonic development. In the first funding period, two transgenic mouse models that enable the identification of GC-B-positive cells by a LacZ reporter or activation of a conditional reporter have been established. It was demonstrated that a cGMP pathway similar to that in DRG neurons also regulates the bifurcation of cranial sensory neurons (Ter-Avetisyan et al. 2014). By analysing the LacZ reporter mice generated by project 6, an interesting spatiotemporal distribution of CNP and its GC-B receptor in the hippocampus, a brain structure involved in memory formation and adult neurogenesis, was identified. In the second funding period, project 6 will, together with other FOR 2060 members, investigate the roles of CNP-induced cGMP signalling in neural differentiation and functional integration of neurons within neuronal networks. It will focus on neuronal populations that express CNP and GC-B in the hippocampus, and on the behavioural impact of GC-B deficiency in DRG neurons and the dentate gyrus using a conditional mouse model for GC-B.
There is accumulating evidence that cGMP production in the nociceptive system contributes to pain processing and axonal regeneration during peripheral neuropathy, a condition where damage resulting from mechanical or pathological mechanisms is inflicted on nerves within the peripheral nervous system (PNS). Previous work of the new project 10 that was performed in collaboration with FOR 2060 members (e.g., with Friebe, project 3; Lukowski/Ruth, project 5; Schmidt, project 6) indicates that pain-relevant cGMP production in the PNS depends on both NO-GC and pGCs, and that distinct K+ channels and CRP4 might be downstream effectors of cGMP and cGKI in sensory neurons (Lu et al. 2015; Lu et al. 2014; Schmidtko et al. 2008a; Schmidtko et al. 2008b). Based on these data, the project will use conditional mouse mutants provided by FOR 2060 investigators to dissect how specific cGMP signalling pathways contribute to pain processing and regeneration after peripheral nerve injury. Project 10 will closely interact with project 6 to study the functional consequences of impaired sensory axon bifurcation as well as a potential novel, bifurcation-independent role of CNP and GC-B in the nociceptive system. Both projects will collaborate with other FOR 2060 projects to analyse cGMP dynamics in sensory neurons under baseline and pathological conditions (with Feil/Feil, project 1 and project Z), to further investigate the functions of NO-GC in the nervous system (with Friebe, project 3), to characterize the downstream mechanisms of neuronal cGMP signalling (with Lukowski/Ruth, project 5), and to study the role of cGMP in the auditory system (with Rüttiger/Knipper, project 8).
Project 8 (Rüttiger/Knipper) investigates the role of cGMP for the viability of sensory hair cells involved in hearing. For the hearing organ, the roles of NO-GC and its downstream pathways are currently controversially discussed, and the functions of pGCs in the cochlea are not fully understood. Recent work of project 8 in collaboration with other FOR 2060 investigators has demonstrated the expression and function of PDE5 and cGKI in inner hair cells and auditory neurons of rodents for the first time (Jaumann et al. 2012). Activation of this cGMP cascade resulted in protection from noise-induced hair cell damage and hearing loss. The aim of this project is to clarify the function of cGMP-generating receptors and cGMP effectors in individual cochlear compartments and to provide a basis for the development of new cell protective therapies to prevent noise- and age-related hearing loss. In the first funding period, the analysis of mouse models provided by FOR 2060 members and others revealed differential expression patterns and functional roles of cGMP generators (e.g., NO-GC isoforms, GC-A, and GC-B) and downstream mediatiors (e.g., BK channels, IRAG). Based on these results, the project will now focus on the functions of individual NO-GC isoforms and the ANP receptor, GC-A, in the context of normal hearing and noise-induced hearing loss. Because PDE9A might be a druggable target to increase cGMP pools that are predominantly controlled by ANP/GC-A (Lee et al. 2015), the expression of PDE9A in the inner ear and the cytoprotective effect of PDE9 inhibition on noise-induced hair cell loss will be tested. Project 8 collaborates with a number of other FOR 2060 projects that provide mouse models and cGMP imaging technology (e.g., Feil/Feil, project 1 and project Z; Friebe, project 3; Nikolaev, project 4; Lukowski/Ruth, project 5). Moreover, there will be particularly strong interactions with the other two projects that study cGMP signalling in sensory neurons. Here, the consequences of impaired GC-B signalling for the auditory pathway will be analysed (with Schmidt, project 6) and similarities between cGMP signalling pathways in sensory neurons of the auditory and nociceptive system will be characterised (with Schmidtko, project 10).
Finally, project Z (Feil) covers various activities to provide a general framework for successful development of the research unit:
- Coordination, administration, public relations
- Scientific symposia and workshops
- Networking and collaborations
- Imaging platform
- Central mouse breeding unit
- Promotion of gender equality and young researchers